SUMMARY The viral reservoir in latently infected CD4+ T lymphocytes represents the key challenge for achieving an HIV-1 cure. We recently reported that the combination of the broadly neutralizing antibody (bNAb) PGT121 together with a TLR7 agonist resulted in a substantial delay or prevention of viral rebound following ART discontinuation in SHIV-infected rhesus monkeys. However, the biology and heterogeneity of the viral reservoir, as well as the mechanism(s) by which these immunotherapeutic strategies target the reservoir, remain to be determined. We hypothesize that there is substantial heterogeneity in the anatomic locations, cellular phenotypes, host transcriptomes, and viral sequences of the viral reservoir, and that certain subsets of reservoir cells are more susceptible to immune-mediated destruction. An improved understanding of the different cellular subsets that comprise the viral reservoir will be essential for understanding the partial efficacy of current interventions and for developing improved next-generation HIV-1 cure strategies. In this proposal, we will continue an ongoing collaboration between Alex K. Shalek at Massachusetts Institute of Technology (MIT), who is an expert in single-cell genomic profiling approaches, and Dan H. Barouch at Beth Israel Deaconess Medical School (BIDMC), who is an expert in conducting HIV-1 cure studies in rhesus monkeys and humans. This unique and synergistic partnership will apply the most insightful, cutting-edge, single-cell `-omics' assays to the most relevant blood and tissue specimens from both monkeys and humans, leading to a deeper understanding of the viral reservoir. Overall, we will utilize these cutting-edge, single-cell `-omics' technologies to define, at multiple levels, the functional heterogeneity of the viral reservoir and to evaluate the mechanism by which current immunotherapeutic interventions target subsets of replication-competent reservoir cells. Such knowledge will be leveraged to define the susceptibility of reservoir subpopulations to immune-mediated destruction by comparing reservoir identity and phenotype following distinct immunotherapy protocols, and to refine and develop next generation HIV-1 cures. To accomplish this goal, we propose the following two Specific Aims: Aim 1: To evaluate the anatomic, cellular, molecular, and viral heterogeneity of the viral reservoir in ART- suppressed rhesus monkeys using single-cell and population genomic profiling strategies Aim 2: To determine the extent to which current immunotherapeutic interventions target various subpopulations of the viral reservoir in rhesus monkeys